Method of fixing nitrogen utilizing mycobacterium butanitrificans



United States Patent 3,390,975 METHOD OF FIXING NITROGEN UTILIZING MYCOBACTERIUM BUTANITRIFICANS Vernon F. Coty, Trenton, N..I., and John B. Davis, Dallas,

Tex., assignors to Mobil Oil Corporation, a corporation of New York No Drawing. Filed Mar. 8, 1965, Ser. No. 438,087

8 Claims. (Cl. 717) ABSTRACT OF THE DISCLOSURE New microbes, Mycobacterium butanitrificans, Mycobacterium parajfinicum and Mycobacterium phlei, are disclosed which have the unusual property of fixing nitrogen without the need of carbohydrates and using hydrocarbons as an energy source.

This invention relates to nitrogen fixation. More particularly, it relates to the conversion of nitrogen into organic compounds by the action of certain microbes on hydrocarbons in the presence of elemental nitrogen.

The fixation of atmospheric or gaseous nitrogen by certain microorganisms is known. For example, aerobic bacteria of the genus Azotobacter, certain anaerobic bacteria, particularly the Clostridium pasteurianum, the Rhizobia in the root nodules of the legumes and certain blue-green algae have been demonstrated to convert nitrogen into microbial protein nitrogen. While recent work has shown the cultivation of hydrocarbon oxidizing microbes in the presence of gaseous, elemental nitrogen but in the absence of other nitrogen compounds, it is desired to extend the applicability of such a process to other hydrocarbons and to other microorganisms.

The Northern Utilization Research and Development Division of the US. Department of Agriculture, Peoria, 111., has designated Mycobacterium butanitrificans as follows: NRRL B3168.

Accordingly, it is an objective of this invention to provide a process in which certain hydrocarbons can be oxidized with accompanying fixation of nitrogen. Another aim is the provision of a microorganism capable of growing on such hydrocarbons and on elemental nitrogen as the only extraneous nitrogen source. These and other purposes appear in the following description and examples, not limitative, but given for illustrative purposes only.

The goals of this invention are accomplished by the finding of new species of microorganisms which has been named Mycobacterium butanitrificans, n. sp. This organism, the cells of which have a rough, waxy surface, grows well in the presence of certain hydrocarbons with gaseous, elemental nitrogen as its only source of nitrogen for conversion to organic nitrogen compounds within its cells or extra-cellularly. The fixation of elemental nitrogen occurs smoothly as the microorganism, contained in an oxidator, is fed oxygen, a hydrocarbon such as n butane, and nitrogen admixed with oxygen or fed separately. A healthly cellular growth and division occurs and nitrogen compounds are produced. These appear mainly as part of the microbial protein or other organic materials. These may be used separately or combined as fertilizers. Mycobacterium has never been reported to fix nitrogen. Hence, the action of the microorganism of this invention is surprising and even more so when the nitrogen fixation is coupled with the metabolism of the unconventional substrate, the hydrocarbon.

Fro-m the above it can readily be appreciated that the processes involved are chemical processes or manners of new manufacture requiring an operator who maintains appropriate conditions and drives the microorganisms to the desired results. The operator separates the result- "ice ant mixtures or the desired products for further utilization as desired.

The invention will be further understood by reference to the examples and description below. In the examples, parts and percentages given are by weight unless otherwise noted.

Example I A number of plates were prepared using a nitrogendeficient mineral agar. These were seeded with bacteria present in an oil-contaminated soil. Each was then incubated for about 2 months at 30 C. In an atmosphere of air and n butane (30% by volume). Examination of the plates was 'made periodically and after the stated time one plate contained a rough, waxy, yellow to orange growth. The growth and overgrowth was examined and was found to contain microbial cells substantially all of which were alike.

Some of the cells were examined under the microscope for characterization. They were found to be non-motile. The cells were acid-fast, stainable rods and, of course, aerobic. While they varied somewhat in size and pigmentation, they were all substantially about 0.3 to 0.6 by 0.5 to 4 microns in size. They were thus identified as a member of the genus, Mycobacterinm.

Transfers of small amounts of the active cells were made to several other of nitrogen-deficient agar plates, and these were incubated as before. In each instance a strong growth of the acting microbe resulted. Based on this, it was concluded that an organism had been found which fixed atmospheric nitrogen while metabolizing n-butane.

Example II An oxidator was charged with 3 liters of an aqueous substrate containing the following salts, in the amounts given in parenthese being gram/liter: Na HPO (0.3), KH PO (0.2), MgSO -7H O (0.1), FeSO -7H O (0.005) and NaMoO 2H O (0.002). These salts are typical of those used in standard aqueous nutrients used in microbial growth, the entire aqueous substrate being, of course, free of nitrogen. To the substrate was added a small inoculum of the microbes from one of the transfer plates prepared in Example I.

The composite was then stirred while air containing n-butane vapors was fed into the stirred mixture. The gas stream coming from the oxidator was discarded, through in other instances it is recycled or passed to another oxidator or to traps for recovery of the hydrocarbon. The composite was kept at room temperature, being 25 C.30' C., and the flow rate of the gaseous stream was abaut 0.5 liter per minute. Turbidity appeared quite rapidly and growth was good. The yields of cells was 1.29 g. or 0.43 gram per liter. The cells contained 27% protein and 19% liquids.

The usual standard of proof for nitrogen fixation was applied and was found to have been exceeded by 2 to 3 times, since 19 micrograms of fixed nitrogen per ml. of solution was evident.

The polymer fraction in the liquid part amounted to 30%. In the triglyceride fraction only palmitic acid was found in this experiment.

In the absence of the elemental nitrogen little or no growth resulted.

To demonstrate the applicability of this invention to other hydrocarbons, the experiment in Example II may be repeated using a n-butylbenzene. Recycling is used with addition of n-butylbenzene to the reentry stream periodically to keep an adequate food source circulating. Vary good growth with nitrogen-fixation is attained.

In another run using ethane similar results are obtained.

Usually, it is desired to increase rates of production, so to demonstrate that the microorganism of this invention may grow and accomplish the objectives of this invention under violent agitation, the oxidator used in Example 11 is equipped wih a sparger and an impeller, there being also present shearing blades. The gaseous mixture is expelled through the sparger, and the stirrer is used to force the mass against the shearing surfaces. Since many of the cyclic hydrocarbons are solids or wellbodied liquids, it is desired to prevent them from occluding the microbial cells. This is accomplished by imparting a violent agitation to the mixture of gas, liquid and solids to the point that the size of the hydrocarbon particles is reduced to microns or less. The substrate used is n-octadecane. Using the aqueous nutrient described in and along with the other conditions of Example II but violently agitating the mass (impeller speeds of about 1800 rpm.) to effect the said size reduction, it is noted that microbial growth not only occurs but is much faster, and the yield of organic nitrogen compounds is substantially increased.

Example III A small amount of the microbe was taken from one of the transfer plates and inoculated into 10 ml. of aqueous medium having the following compositions: MgSO .7H O (0.2), Na HPO (0.3), KH PO (0.2), CaCl (0.01), Na CO (0.1), FeSO .7H O (0.005), MnSO, (0.002) and Na MoO .2H O (0.002). A control was not inoculated.

Both systems were gassed with a mixture of air/nbutane (70%30%) attached to manometers and incubated. The gas uptake was measured periodically. After 18 days there was a 57 mm. change (gas uptake) in the system containing the organism of this invention as compared to no change in the control. Also, the control did not show the turbidity increase that was attained in the inoculated flask. The bacterial growth was a proliferation of the said Mycobacterium butanitrificans.

The hydrocarbons that can be used in this invention include aliphatics and aromatics. Included among the numerous hydrocarbons that are metabolized by the new species are methane, ethane, n-propane, n-butane, isobutane, the various pent-anes, the various hexanes and higher aliphatics such as dodecane and tetradecane. Kerosene may be used and oily products such as mineral oils and paraffin, petroleum mulches, petroleum resins, asphalt, among others, may also be employed. When using the aromatics, it is preferred that the aromatic ring carry on it alkyl substituents having 4 carbons or more. Therefore, such compounds as butyl'benzene, the isomeric butylbenzenes, the isomeric amylbenzenes, the isomeric hexylbenzenes, the isomeric heptylbenzenes, and the isomeric octylbenzenes are the preferred aromatics. Of course other alkyl substituents may be present also, and various mixtures of any of the hydrocarbons, mentioned above or below, may be used.

Among the naphthenes which may be used are methylcyclopentane, the dimethylcyclopentanes, the trimethylcyclopentanes, ethylcyclopentane, the diethylcyclopentanes, the isomeric propylcyclopentanes, the isomeric butylcyclopentanes, the isomeric amylcyclopentanes, the isomeric hexylcyclopentanes, the isomeric heptylcyclopentanes, and the isomeric octylcyclopentanes. Also included among these naphthenes are methylcyclohexane, the dimethylcyclohexanes, the trimethylcyclohexanes, the tetramethylcyclohexanes, ethylcyclohexane, the isomeric propylcyclohexanes, isopropyl-4-methylcyclohexane, the isomeric butylcyclohexanes, the isomeric amylcyclohexanes, the isomeric hexylcyclohexanes, the isomeric heptylcyclohexanes, and the isomeric octylcyclohexanes.

The fermentation reaction mixture should also contain, in accordance with conventional practice in carrying out microbiological reactions, mineral salts for the growth of the oxidative microorganism. These salts should furnish potassium, ferrous or ferric, calcium, magnesium, phosph ate and sulfate ions, as well as ions of tract elements such as zinc, manganese, copper, and molybdenum.

The fermentation reaction mixture, during the oxidative reaction, is maintained under conditions to insure optimum growth of the fermentation microorganism. The temperature, for example, should be maintained between about 20 and about 55 C. Preferably, the temperature should be maintained in the neighborhood of 30 C. Further, the pH of the reaction mixture should be maintained near neutrality, namely, at about 7.0. However, the fermentation reaction may be carried out at a pH between about 5.5 and 8.5.

The fermentation reaction mixture will consist primarily of water. The water may constitute 99%, or more, by weight of the liquid phase of the fermentation reaction mixture. However, the water may consitute a much lesser portion of the fermentation reaction mixture. For example, the fermentation reaction mixture may contain as little as 50% by weight of water. Generally, any proportion of water heretofore employed in microbial oxidation of hydrocarbons may be used.

The microbial oxidation reaction requires that oxygen be supplied to the fermentation reaction mixture. Oxygen can be supplied to the fermentation reaction mixture by employing reactors open to the atmosphere. With agitation of the reaction mixture, the surface thereof exposed to the atmosphere is continuously being renewed and oxygen is thereby taken up by the mixture. If desired, oxygen may be supplied by bubbling oxygen, or any oxygen-containing gas such as air, through the fermenta tion reaction mixture. Further, if desired, for the purpose of avoiding excessive evaporation of water from the fermentation reaction mixture, the oxygen or oxygencontaining gas may be humidified prior to bubbling through the fermentation reaction mixture. It will be realized that, where oxygen or oxygen-containing gas is bubbled through the reaction mixture, the bubbling of the gas may provide the desired agitation of the reaction mixture. On the other hand, agitation by other means may additionally be employed in order to insure that the agitation is adequate.

Following the fermentation reaction, the various oxidation products of the hydrocarbons are removed by conventional methods from the fermentation reaction mixture. The fermentation reaction mixture may be subjected to such procedures as may be required to remove the body cells of the microorganism. Suitable procedures include decantation, centrifuging, and filtration. The mixture may also be subjected to such procedures as may be required to remove any remaining hydrocarbon. Extraction of the reaction mixture with a solvent, such as petroleum ether, in which the hydrocarbon, but not certain oxidation products, is soluble may be employed. Thereafter the reaction mixture may be extracted with a solvent in which said oxidation products are soluble. With water contained in the reaction mixture, this solvent will be a waterimmiscible solvent. Satisfactory results have been obtained by extracting the fermentation reaction mixture with diethylether. Some oxidation products may then be recovered from the diethylether or other solvent employed by evaporation or other suitable procedure. Also, oxidation products may also be recovered from the reaction mixture by steam distillation. It is also possible to use the entire aqueous mass as a fertilizer or to remove the water from it and use the residue as a fertilizer.

In any of the processes it is preferred to keep the pH neutral. Thus, while the pH may be about 6.0 to about 8.0, it is preferred to keep it at 7.0. The phosphates, such as potassium dihydrogen phosphate, are used as buffers to do this. The fermentors can be run on a continuous basis, the cells or products being removed while leaving some cells as an inoculate and while adding fresh medium.

When gases or vapors are being used as the substrates, the concentration of the hydrocarbon is not critical. Generally, it is 15% to 50% of the hydrocarbon/air mixture. While oxygen is required for the oxidation no special means for supplying it are needed. Thus, the fermentor may simply get its oxygen and the nitrogen to be fixed rnerely from the surrounding air by normal exposure of the mass to the air. Usually, it is prefer-red to feed air along with the hydrocarbon since this leads to greater growth by increased contact of the cells with the feed.

When liquid hydrocarbons are being fed, it is preferred to use lower concentrations and this is also true for the solids. The concentrations are about 0.1% to about 1.0% based upon the weight of the medium. While concentrations may be increased by using stepwise additions and vigorous agitation, the organisms grow very well in the absence of such steps, and accordingly, normal conditions are usually used.

To demonstrate the applicability of the principles of this invention to other Mycobacterium two additional species are used in the above manners. These are Mycobacterium paraflinicum and Mycobacterium phlei, old species, but new in nitrogen fixation. The processes and compositions of this invention include these organisms. Since these organisms are readily available, they are of special interest in the fertilizer applications of this invention which are described above and further below.

The process of this invention is useful in the production of an organic fertilizer. The cells and other products produced can be added to soil or the hydrocarbon can be fed to soils to which have been added Mycobacterium butanitrificans, Mycobacterium parafiinicwm and/or Mycobacterium phlei, each separately or in admixture with themselves or other microorganisms. In this procedure, the hydrocarbons are generally added in amounts about 0.01% to about 0.1% by weight of the soil, but the exact amount will vary depending upon soil, weather and similar conditions. Many soils are very low in nitrogen content, and a process of this invention is directed to increasing soil nitrogen. Thus, one will seek to create a growth situation by adding a relatively large amount of a given hydrocarbon to the soil or by adding a relatively large amount of microbial inoculum or by adding both. In this connection it is to be appreciated that cellular matter added to the soil may contain dead or live cells or mixtures thereof and the soil additive may include the culture solution or What remains of it upon evaporation of the water for the media usually contain nitrogenous matter derived from autolyzed cells or possible extracellular products in addition to salts purposely present. The fixed nitrogen in the solution usually represents about of the total amount fixed. Further, carbohydrates and lipids are also produced. These as well as the protein material, can be used as fertilizers to add nitrogen to the ground by adding them to soils containing conventional nitrogen fixers that feed on conventional substrates.

The microorganism and compositions containing cells thereof and the processes of this invention are particularly useful in petroleum mulches. It is known that the laying down of a petroleum coating, such as asphalt, on the ground keeps moisture within the ground beneath the coating. Such coatings are placed over seed beds leaving the areas between seed rows exposed to receive normal rainfall. Water moves laterally from the unseeded area to contiguous seeded, covered areas and is held there by the coating and is available for use by the seed or the plant therefrom for longer than normal time periods. The coating is of such a nature that it can be pierced by the germinating seed and growing plant. Such mulches are used also with means to divert run-oh water from seeded contributing areas to seeded benched areas which means may include confining means other than petroleum mulches. Applying the principles of this invention to such mulches comprises applying to the soil the Mycobacterium of the invention plus the hydrocarbonaceous substrate on which they feed. While the organism of this invention is able to feed directly on a given petroleum product, such as a petroleum mulch, a petroleum resin, or asphalt, it is beneficial to add hydrocarbons to the ground or to the petroleum coating, preferably the former. By these procedures, mulches of enhanced value are attained, since they provide not only for water retention but for nitrogen fixation. Land in arid regions is generally very low in nitrogen content. Such land may be reclaimed by use of the processes of this invention.

While this invention has been disclosed herein in connection with certain embodiments and certain procedural details, it is clear that changes, modifications or equivalents can be used by those skilled in the art; accordingly, such changes within the principles of this invention are intended to be included within the scope of the claims below.

We claim:

1. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Mycobacterium. butanitrificans; cultivating said cells to produce a culture thereof; forming an aqueous mixture that is a nutrient for microorganisms and that is substantially nitrogen-free; placing said aqueous mixture into a vessel; bringing elemental nitrogen, oxygen, a hydrocarbon and some of said cells of Mycobacterium butanitrificans into contact with each other in said aqueous mixture; and allowing said cells to metabolize said bydrocarbon and nitrogen, said Mycobacterium butanitrificans being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL B-3168.

2. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Mycobacterium butanitrificans; cultivating said cells to produce a culture thereof; admixing in a vessel a hydrocarbon, elemental nitrogen, oxygen and some of said cells of Mycobacterium butanitrificans; and allowing said organism to grow on said hydrocarbon with said elemental nitrogen as its only source of nitrogen for growth, thereby fixing said elemental nitrogen, said Mycobacterium butanitrificans being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL B- 3168.

3. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Mycobacterium butanitrificans; cultivating said cells to produce a culture thereof; forming in a vessel a mixture of a hydrocarbon, elemental nitrogen, oxygen and some of said cells of Mycobacterium butanitrificans which cells oxidize said hydrocarbon and fix elemental nitrogen; keeping the resultant mixture at a pH of about 6.0 to about 8.0; and allowing said microorganism to grow on said mixture, thereby fixing said elemental nitrogen, said Mycobacterium butanitrificans being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL B-3l68.

4. A process in accordance with claim 3 which is carried out under normal conditions of temperature and pressure.

5. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Mycobacterium butanitrificans; cultivating said cells to produce a culture thereof; forming in a vessel a mixture of nitrogen, oxygen and a hydrocarbon; feeding said mixture to some of said cells of Mycobacterium butanitrificans and allowing said cells to metabolize said hydrocarbon and nitrogen, said Mycobacterium butanitrificans being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL B-3l68.

6. A process in accordance with claim 5 in which the cells are in the presence of an aqueous nutrient during said feeding and said metabolization.

7. A process in accordance with claim 5 in which said nitrogen and said oxygen are fed together as air.

8. A process for increasing soil nitrogen which comprises isolating from soil cells of a microorganism now known as Mycobacterium butanitrificans; cultivating said cells to produce a culture thereof; adding a hydrocarbon and some of said cells of Mycobacterium butanitrificans to a second soil; exposing the resultant modified soil to nitrogen; and effecting fixation of the elemental nitrogen by allowing said organism to grow on said hydrocarbon, oxygen and nitrogen, thereby converting said nitrogen into compounds said M ycobacterium butanitrifieans being numbered by the US Agricultural Research Laboratory as follows: NRRL B3168.

References Cited UNITED STATES PATENTS 3,210,179 10/1965 Davis et a1. 71-7 5 DONALL H. SYLVESTER, Primary Examiner.

R. BAJEFSKY, Assistant Examiner. 

